Multiple bacterial pathogens have evolved mechanisms that engage intracellular signaling pathways in the host cell to achieve successful infection (Zaharik et al, Int. J. Med. Microbiol. 291:593-603 (2002)). An example of such a pathogen is the Gram negative bacteria Shigella flexneri, the etiologic agent for the diarrheal disease shigellosis (Sansonetti, Am. J. Physiol. Gastrointest. Liver Physiol. 280:G319-323 (2001)). A key step in the pathogenesis of shigellosis is the ability of the bacteria to enter the normally non-phagocytic cells of the colonic mucosa. At the site of Shigella entry, the host actin cytoskeleton undergoes dramatic changes, including the formation of filopodia and lamellipodia, which are subsequently organized into long, actin-rich extensions that engulf the invading bacterium (Adam et al, J. Cell Biol. 129:367-381 (1995)). The changes in the actin cytoskeleton observed during Shigella infection are mediated by bacterial effectors that are part of a Type III Secretion System (TTSS) that is activated following contact between the bacterium and the host cells. The TTSS inserts a pore complex, comprised of Shigella proteins IpaB and IpaC, into the host cell plasma membrane that allows for delivery of other bacterial effector proteins into the host cell (Zaharik et al, Int. J. Med. Microbiol. 291:593-603 (2002), Blocker et al, J. Cell Biol. 147:683-693 (1999), Tran Van Nhieu et al, EMBO J. 16:2717-2729 (1997), Niebuhr et al, Mol. Microbiol. 38:8-19 (2000)). A key event during the initial phase of infection is the induction of actin polymerization at the site of Shigella contact with the host cell membrane, which results in massive cytoskeletal rearrangements, and the formation of actin foci at the site of the invading bacterium (Adam et al, J. Cell Biol. 129:367-381 (1995)). The insertion of Shigella IpaC into the membrane results in changes in the actin cytoskeleton, characteristic of the activation of the Rho family GTPases Cdc42 and Rac (Tran Van Nhieu et al, EMBO J. 18:3249-3262 (1999)). Both Cdc42 and Rac localize to the site of bacterial entry, and their activation has been shown to be required for efficient uptake of Shigella (Mounier et al, J. Cell Sci. 112:2069-2080 (1999), Shibata et al, Curr. Biol. 12:341-345 (2002)). The tyrosine kinase Src is also translocated to the site of the invading bacterium, and is thought to act as both a positive and negative regulator of the entry process. Src exerts its positive role by promoting the formation of actin foci, but it also acts negatively to down-regulate Rho (Dumenil et al, J. Cell Biol 143:1003-1012 (1998), Dumenil et al, J. Cell Sci. 113:71-80 (2000)). However, the role of tyrosine phosphorylation in the uptake of Shigella flexneri has not been fully explored, and the link between tyrosine kinases and Rho GTPase-dependent actin polymerization during this process has yet to be defined.
The Abl tyrosine kinase has been shown to regulate Rac-dependent cytoskeletal dynamics in mammalian cells, suggesting that Abl kinases may play a role in bacterial uptake (Plattner et al, Genes Dev. 13:2400-2411 (1999)). The mammalian Abl family of tyrosine kinases is comprised of Abl and Arg (Abl2), and has been implicated in the regulation of cell proliferation, survival, adhesion, and migration (Pendergast, Adv. Cancer Res. 85:51-1-(2002)). While the functions of the constitutively active chimeric oncogene Bcr-Abl have been well described, the cellular functions of Abl and Arg have remained elusive. Genetic studies have implicated Abl and Arg in the regulation of cytoskeletal dynamics. Drosophila melanogaster that lack Abl exhibit defects in growth cone motility, axon guidance, and epithelial cell polarity (Pendergast, Adv. Cancer Res. 85:51-1-(2002)). The defective growth cone phenotype is identical to that of Drosophila lacking profilin, a protein known to be involved in cytoskeletal dynamics (Wills et al, Neuron 22:291-299 (1999)). A similar phenotype is observed in flies expressing dominant negative Cdc42, or mutants of Trio, a Guanine Nucleotide Exchange Factor (GEF) for Rac and Rho (Wills et al, Neuron 22:291-299 (1999), Liebl et al, Neuron 26:107-118 (2000), Bateman et al, Neuron. 26:93-106 (2000)). Since Rho family GTPases have been shown to regulate the formation of F-actin structures such as filopodia and lamellipodia, these observations suggest that Drosophila Abl may regulate cytoskeletal re-organization and cell motility. Mice lacking Abl and Arg also exhibit cytoskeletal defects, resulting in delayed closure of the neural tube, and death before embryonic day 11 (Koleske et al, Neuron. 21:1259-1272 (1998)). Normal neuroepithelium display an ordered pattern of actin filaments at their apical surface, where Abl and Arg are normally located. In the Abl/Arg null mice, this apical actin latticework pattern is absent, and unorganized bundles of actin filaments are found at the basolateral surface of the cell (Koleske et al, Neuron. 21:1259-1272 (1998)). Moreover, it has been shown that Abl is required for formation of Rac-dependent lamellipodia in response to PDGF (Plattner et al, Genes Dev. 13:2400-2411 (1999)). These properties of the normal Abl family tyrosine kinases are consistent with the observed changes in the actin cytoskeleton of Bcr-Abl-expressing cells. Expression of Bcr-Abl induces the formation of filopodia and lamellipodia, and extension of pseudopods onto a fibronectin matrix (Salgia et al, J. Clin. Invest. 100:46-57 (1997)). These cytoskeletal effects were found to be a result of the increased tyrosine kinase activity of Bcr-Abl, and were reversed in the presence of Abl kinase inhibitors (Gaston et al, Exp. Hematol. 28:351 (2000)). These studies demonstrate that the Abl family kinases regulate cytoskeletal dynamics. Furthermore, Abl and Arg are unique among all known tyrosine kinases in that they contain a carboxy-terminal actin binding domain, and have been shown to have actin bundling activity (Pendergast, Adv. Cancer Res. 85:51-100 (2002)). Altogether, the Abl kinases are uniquely suited to link extracellular stimuli, such as infection by bacterial pathogens, to reorganization of the actin cytoskeleton.
The present invention results, at least in part, from studies, demonstrating a requirement for Abl and Arg in Shigella flexneri infection, and linking the requirement for Abl kinase activity to the Rho family GTPases Cdc42 and Rac during bacterial uptake.